Figure 4.

NI rats exhibit increased surface antigen expression of CD11b on isolated HP microglia. a, Adult rat HP were microdissected following cold saline perfusion and brought to single-cell suspensions using Miltenyi's Neural Dissociation Kit followed by myelin depletion. Myelin-depleted cells were stained with APC-conjugated CD11b and analyzed using flow cytometry. Representative presorted populations are shown as distinct peaks. An unstained control is overlaid for reference (shaded histogram). b, Myelin-depleted cells from rats in each neonatal treatment group were stained with APC-conjugated CD11b to assess expression on microglia independent of selection using magnetic beads, which could influence antigen expression. The bar graph shows greater average (±SEM) MFI for CD11b reactivity from NI (n = 10) compared with PBS (n = 9) rats (*p < 0.05). c, Myelin-depleted cells from rats in each neonatal treatment group were sorted into CD11b⁺ and CD11b⁻ fractions using magnetic beads. Representative postsorted (MACS) populations are shown. Purity was consistently >93% CD11b⁺ and ∼99% CD11b⁻ for all samples. A rightward shift can be seen in the population of CD11b⁺ cells from NI rats, indicating increased expression (vertical line is provided for comparison). d, Sorted CD11b⁺ cells were stained with APC-conjugated CD11b/c and CD45 (a marker highly expressed on infiltrating macrophages), which revealed a large, dense cluster of CD11b^high/CD45^low cells. In contrast, CD11b^high cells that also stained brightly for CD45 accounted for only 4.2% of the population, indicating the perivascular macrophage population was minimal.